Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by, for example, thrombocytopenia, hemolytic anemia and perturbing neurological dysfunction. It was formerly a poor prognostic disease with approximately 80% of patients died within 3 months. However, currently, the prognosis has considerably improved by plasma exchange.
Recently, it was reported that TTP might be attributed to the decrease in activity of VWF-cleaving enzyme (ADAMTS13). Namely, it has been revealed that an IgG type inhibitor against VWF-cleaving enzyme is produced to decrease the enzyme activity, causing the acquired TTP (Non-patent Document Nos. 1 and 2). Further, Upshaw-Schulman syndrome (USS) which is congenital TTP was proved to be genetically deficient in VWF-cleaving enzyme (Non-patent Document No. 3). The gene encoding this VWF-cleaving enzyme was proved to be ADAMTS13 (Non-patent Document Nos. 4 and 5).
ADAMTS13 is a zinc metalloprotease and specifically cleaves VWF subunit at Tyr842-Met843 bond. The activity of this enzyme is measured by VWF multimer analysis in which VWF is employed as a substrate to detect the produced VWF fragments by electrophoresis. Since this method has a merit to allow accurate measurement of ADMATS13 activity, but is accompanied with complicated operation, it has been desired to develop an especially simpler measuring method.
Non-patent Documents Nos. 6 to 8 report a method wherein the A2 domain in VWF or a fraction thereof is used a substrate for ADAMTS13 to measure ADAMTS13 activity. They are not natural substrates, but expressed in E. coli by genetic recombination. Based on the fact that these substrates are degraded by ADAMTS13 present in plasma samples, the measuring methods described above detect these substrates in molecular weight by electrophoresis and Western blotting, and then react these substrates with the enzyme to measure immunologically the remaining undegraded substrates, thereby to detect and determine ADAMTS13 activity. However, since these methods use a reverse correlation between the ADAMTS13 activity and the observed signal intensity with the standard curve taking a negative gradient, a problem remains to be solved that these method are not able to provide satisfactory sensitivity and reproducibility within a clinically important region of as low as 5% or below.
To improve this problem, a method for measuring ADAMTS13 activity of plasma using a quenchable fluorescent substrate has been reported (Non-patent Document No. 9). This method contains a standard curve taking a positive gradient where fluorescence intensity increases as ADAMTS13 activity increases. However, the method has a problem for use in a general clinical laboratory, because it must use a specially chemically synthesized and expensive substrate to conduct a rate assay by a fluorometer.
[Non-patent Document No. 1] New Engl. J. Med. 339, 1578-1584, 1998
[Non-patent Document No. 2] New Engl. J. Med. 339, 1585-1594, 1988
[Non-patent Document No. 3] J. Hematol. 74, 101-108, 2001
[Non-patent Document No. 4] J. Biochem. 130, 475-480, 2001
[Non-patent Document No. 5] J. Biol. Chem. 276, 41059-41063, 2001
[Non-patent Document No. 6] Blood, 103,607-612, 2004
[Non-patent Document No. 7] J. Thromb. Haemost. 2, 485-491, 2004
[Non-patent Document No. 8] Thromb Haemost. 91, 806-811, 2004
[Non-patent Document No. 9] The Journal of Japanese Society on Thrombosis and Hemostasis, 15,421, 2004